Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNALeu(UUR)) gene

Abstract

The gene encoding mt-tRNA(Leu(UUR)), MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A>G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNA(Leu(UUR)) and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A>G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNA(Leu(UUR)) levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation.

Figure 1

Respiratory chain phenotype of cybrid cell lines. (A) Polarography. Graphs show mean values and standard deviations for oxygen consumption of 143B control cells (white bars, n = 10) and of cybrids containing the 3302A>G mutation at 98% (grey bars, n = 3) in intact cells and permeabilized cells respiring on the indicated substrates. (mal = malate; glu = glutamate). (B) Respiratory chain complex activities. Graphs show activities for SCCR—Complex II+III, QCCR—Complex III, COX—Complex IV and CS as mitochondrial matrix marker. Graphs are mean values and standard deviations for 143B control cells (white bars, n = 5) and cybrids containing the 3302A>G mutation (grey bars, n = 3).

Figure 2

Determination of steady-state levels of tRNA^Leu(UUR) in cybrid clones. (A) Levels of tRNAs analysed by high-resolution PAGE followed by blot hybridization in two control cell lines and the cybrid clone containing 77% of the 3302A>G mutation and 98% of the 3302A>G mutation, as well as a clone containing the 3243A>G mutation (3243 100%). RNA was loaded in two concentrations in order to ensure linearity of the densitometric signal. (B) The densitometric values for 1 μg of 143B RNA were arbitrarily set to 1.0 in order to get an estimate of the variability of the measurements. Upper panel: levels of mt-tRNA^Leu(UUR) (black bars) and tRNA^Val (grey bars) were normalized to nuclear encoded 5S rRNA; lower panel: Levels of mt-tRNA^Leu(UUR) were normalized to tRNA^Val.

Figure 3

Analysis of mitochondrial transcript processing in cybrid clones, human skeletal muscle and primary human myoblasts, myotubes and fibroblasts. Levels of the precursor transcript RNA19 and its processing intermediates as well as ND1 and COX I mRNA analysed by agarose gel electrophoresis followed by blot hybridization. Samples were from two healthy human skeletal muscles (lanes 1 and 3), primary human myoblasts from these biopsies (lanes 4 and 5), primary human myotubes differentiated in vitro from such myoblasts (lanes 6 and 7), primary human fibroblasts (lanes 8 and 9), control cell lines (143B, 3243 wt, lanes 10 and 12) and from the cybrid clone containing 77% of the 3302A>G mutation (lane 13), 98% of the 3302A>G mutation (lane 14), as well as from the clone containing the A3243G mutation (3243 100%, lane 11). RNA from 143B rho⁰ cells was also loaded (lane 15). 5 μg of total RNA was loaded in each lane. Probes used for hybridization are indicated on the right, RNA species identified on the left.

Figure 4

Determination of the half-life of RNA19 and ND1 mRNA in cybrid clones treated with EtBr for up to 8 h. Decay of mitochondrial transcripts was analysed by agarose gel electrophoresis followed by blot hybridization to a probe for ND1 mRNA in isogenic control cells containing 100% wt-mtDNA (3243 wt), in the cybrid clone containing the 3302A>G mutation (3302 98%), as well as in the clone containing the 3243A>G mutation (3243 100%).

Figure 5

Analysis of polyadenylation of RNA19. Polyadenylation of RNA19 was analysed by agarose gel electrophoresis of total RNA (5 μg) and poly(A⁺)-RNA (the total yield from 100 μg of total RNA) in 143B control cells, in the cybrid clone containing the 3243A>G mutation, as well as in the clone containing 98% of the 3302A>G mutation. Probes used for hybridization are indicated on the right, RNA species identified on the left.

Figure 6

Determination of the half-life of mitochondrial tRNAs in cybrid clones treated with EtBr for up to 48 h. (A) Decay of mitochondrial tRNAs was analysed by high-resolution PAGE followed by blot hybridization in 143B control cells, in the cybrid clone containing 98% of the 3302A>G mutation, as well as in the clone containing the 3243A>G mutation (3243 100%). RNA (0.5 μg) was loaded in triplicate per sample in order to minimize data acquisition errors of the signals. (B) Densitometric data for tRNA^Leu(UUR), tRNA^Val and tRNA^Lys were normalized to nuclear encoded 5S rRNA.

Figure 7

Aminoacylation levels in cybrid clones. Degree of aminoacylation of mt-tRNA^Leu(UUR) in 143B cells, in the cybrid clones containing 77 or 98% of the 3302A>G mutation as well as in the clone containing the 3243A>G mutation (3243 100%). RNA was isolated from cells and separated by PAGE under acidic conditions. Deacylated RNA samples of all cell lines and a mixture of acylated and deacylated RNA from 143B cells (first lane) were also loaded in order to facilitate identification of the two species.

Figure 8

Analysis of tRNA structure, length and charge by different PAGE methods and northern blotting. RNA samples isolated under neutral conditions from cell lines in order to split off the amino acid were run together with run-off transcripts with the wt or 3302A>G sequence, phosphorylated (IVT P) at the 5′ end or non-phosphorylated (IVT) under different conditions. Gels were blotted and probed with a probe for tRNA^Leu(UUR). (A) Urea containing, acidic conditions. (B) Urea containing, neutral conditions. (C) Native, neutral conditions.